Effect of cellulose nanocrystals derived from Dunaliella tertiolecta marine green algae residue on crystallization behaviour of poly(lactic acid).

Marine green algae biomass residue (ABR), a waste by-product of Dunaliella tertiolecta, left behind after the extraction of oil from the algal biomass, was utilized for the fabrication of cellulose nanocrystals (CNCs). The fabricated sulphuric acid hydrolysed CNCs had needle-like morphology, with dominant cellulose type I polymorph and a high crystallinity index of 89 %. ICP-MS elemental analysis confirmed the presence of a variety of minerals in the ABR. Washed ABR (WABR)/PLA and CNC/PLA bio-composite films were developed via solvent casting technique with varying bio-filler loadings for comparing their effectiveness on the crystallization behaviour of PLA. FESEM, FTIR, XRD and TGA were used to characterize the bio-fillers. The nucleating and crystallization behaviour of the bio-composite films were confirmed using DSC, SAXS and POM analysis which indicated better effectiveness of CNCs with a significant reduction in cold crystallization temperature, and noteworthy increment in crystallinity and spherulite growth rate.

Cocrystal Solubility Advantage and Dose/Solubility Ratio Diagrams: A Mechanistic Approach To Selecting Additives and Controlling Dissolution-Supersaturation-Precipitation Behavior.

Two of the main questions regarding cocrystal selection and formulation development are whether the will be stable and how fast can it dissolve the drug dose. Dissolving the drug dose may require cocrystals with a high solubility advantage over drug (SA = SCC/SD), but these may have limited potential to sustain drug supersaturation. Thus, we propose a twofold approach to mitigate the risk of drug precipitation by optimizing thermodynamic (SA) and kinetic factors (nucleation inhibitors). This risk can be evaluated by considering the cocrystal SA and drug dose/solubility ratio (D0D = Cdose/SD), which in tandem represent the maximum theoretical supersaturation that a cocrystal may generate, the driving force for drug precipitation, and the potential for dose-/solubility-limited absorption. cocrystals with SA and D0D values above critical supersaturation are prone to rapid precipitation, often negating their utility as a solubility enhancement tool. This work presents a mechanistic approach to controlling the dissolution-supersaturation-precipitation behavior of cocrystal systems, whereby relationships between SA, D0D, and the drug-solubilizing power of surfactants (SPD = SD,T/SD,aq) are used to fine-tune cocrystal-inherent supersaturation by rational additive selection. Experimental results with danazol-vanillin cocrystal demonstrate how SA, D0D, and SPD are key thermodynamic parameters to understanding the kinetic cocrystal behavior and how the risks of cocrystal development may be mitigated through the mechanistic formulation design.

The role of aspartic acid in reducing coral calcification under ocean acidification conditions.

Biomolecules play key roles in regulating the precipitation of CaCO3 biominerals but their response to ocean acidification is poorly understood. We analysed the skeletal intracrystalline amino acids of massive, tropical Porites spp. corals cultured over different seawater pCO2. We find that concentrations of total amino acids, aspartic acid/asparagine (Asx), glutamic acid/glutamine and alanine are positively correlated with seawater pCO2 and inversely correlated with seawater pH. Almost all variance in calcification rates between corals can be explained by changes in the skeletal total amino acid, Asx, serine and alanine concentrations combined with the calcification media pH (a likely indicator of the dissolved inorganic carbon available to support calcification). We show that aspartic acid inhibits aragonite precipitation from seawater in vitro, at the pH, saturation state and approximate aspartic acid concentrations inferred to occur at the coral calcification site. Reducing seawater saturation state and increasing [aspartic acid], as occurs in some corals at high pCO2, both serve to increase the degree of inhibition, indicating that biomolecules may contribute to reduced coral calcification rates under ocean acidification.

High throughput solubility and redissolution screening for antibody purification via combined PEG and zinc chloride precipitation.

As upstream product titers increase, the downstream chromatographic capture step has become a significant "downstream bottleneck." Precipitation becomes more attractive under these conditions as the supersaturation driving force increases with the ever-increasing titer. In this study, two precipitating reagents with orthogonal mechanisms, polyethylene glycol (PEG) as a volume excluder and zinc chloride (ZnCl2 ) as a cross linker, were examined as precipitants for two monoclonal antibodies (mAbs), one stable and the other aggregation-prone, in purified drug substance and harvested cell culture fluid forms. Manual batch solubility and redissolution experiments were performed as scouting experiments. A high throughput (HTP) liquid handling system was used to investigate the design space as fully as possible while reducing time, labor, and material requirements. Precipitation and redissolution were studied by systematically varying the concentrations of PEG and ZnCl2 to identify combinations that resulted in high yield and good quality for the stable mAb; PEG concentrations in the range 7-7.5 wt/vol% together with 10 mM ZnCl2 gave a yield of 97% and monomer contents of about 93%. While yield for the unstable mAb was high, quality was not acceptable. Performance at selected conditions was further corroborated for the stable mAb using a continuous tubular precipitation reactor at the laboratory scale. The HTP automation system was a powerful tool for locating desired (customized) conditions for antibodies of different physicochemical properties.

Formation of α-synuclein aggregates in aqueous ethylammonium nitrate solutions.

The effect of adding ethylammonium nitrate (EAN), which is an ionic liquid (IL), on the aggregate formation of α-synuclein (α-Syn) in aqueous solution has been investigated. FTIR and Raman spectroscopy were used to investigate changes in the secondary structure of α-Syn and in the states of water molecules and EAN. The results presented here show that the addition of EAN to α-Syn causes the formation of an intermolecular ß-sheet structure in the following manner: native disordered state → polyproline II (PPII)-helix → intermolecular ß-sheet (α-Syn amyloid-like aggregates: α-SynA). Although cations and anions of EAN play roles in masking the charged side chains and PPII-helix-forming ability involved in the formation of α-SynA, water molecules are not directly related to its formation. We conclude that EAN-induced α-Syn amyloid-like aggregates form at hydrophobic associations in the middle of the molecules after masking the charged side chains at the N- and C-terminals of α-Syn.

Transfection of Mammalian Cells with Calcium Phosphate-DNA Coprecipitates.

Biochemical methods of transfection, including calcium phosphate-mediated and diethylaminoethyl (DEAE)-dextran-mediated transfection, have been used for many years to deliver nucleic acids into cultured cells. Here, we briefly review the use of calcium phosphate-DNA coprecipitates for transfection.

3D plotting in the preparation of newberyite, struvite, and brushite porous scaffolds: using magnesium oxide as a starting material.

Calcium phosphate (CaP)-containing materials, such as hydroxyapatite and brushite, are well studied bone grafting materials owing to their similar chemical compositions to the mineral phase of natural bone and kidney calculi. In recent studies, magnesium phosphate (MgP)-containing compounds, such as newberyite and struvite, have shown promise as alternatives to CaP. However, the different ways in degradation and release of Mg2+ and Ca2+ ions in vitro may affect the biocompatibility of CaP and MgP-containing compounds. In the present paper, newberyite, struvite, and brushite 3D porous structures were constructed by 3D-plotting combining with a two-step cementation process, using magnesium oxide (MgO) as a starting material. Briefly, 3D porous green bodies fabricated by 3D-plotting were soaked in (NH4)2HPO4 solution to form semi-manufactured 3D porous structures. These structures were then soaked in different phosphate solutions to translate the structures into newberyite, struvite, and brushite porous scaffolds. Powder X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive spectrometry (EDS) were used to characterize the phases, morphologies, and compositions of the 3D porous scaffolds. The porosity, compressive strength, in vitro degradation and cytotoxicity on MC3T3-E1 osteoblast cells were assessed as well. The results showed that extracts obtained from immersing scaffolds in alpha-modified essential media induced minimal cytotoxicity and the cells could be attached merely onto newberyite and brushite scaffolds. Newberyite and brushite scaffolds produced through our 3D-plotting and two-step cementation process showed the sustained in vitro degradation and excellent biocompatibility, which could be used as scaffolds for the bone tissue engineering.

Improved Prediction of in Vivo Supersaturation and Precipitation of Poorly Soluble Weakly Basic Drugs Using a Biorelevant Bicarbonate Buffer in a Gastrointestinal Transfer Model.

The characterization of intestinal dissolution of poorly soluble drugs represents a key task during the development of both new drug candidates and drug products. The bicarbonate buffer is considered as the most biorelevant buffer for simulating intestinal conditions. However, because of its complex nature, being the volatility of CO2, it has only been rarely used in the past. The aim of this study was to investigate the effect of a biorelevant bicarbonate buffer on intestinal supersaturation and precipitation of poorly soluble drugs using a gastrointestinal (GI) transfer model. Therefore, the results of ketoconazole, pazopanib, and lapatinib transfer model experiments using FaSSIFbicarbonate were compared with the results obtained using standard FaSSIFphosphate. Additionally, the effect of hydroxypropyl methylcellulose acetate succinate (HPMCAS) as a precipitation inhibitor was investigated in both buffer systems and compared to rat pharmacokinetic (PK) studies with and without coadministration of HPMCAS as a precipitation inhibitor. While HPMCAS was found to be an effective precipitation inhibitor for all drugs in FaSSIFphosphate, the effect in FaSSIFbicarbonate was much less pronounced. The PK studies revealed that HPMCAS did not increase the exposure of any of the model compounds significantly, indicating that the transfer model employing bicarbonate-buffered FaSSIF has a better predictive power compared to the model using phosphate-buffered FaSSIF. Hence, the application of a bicarbonate buffer in a transfer model set-up represents a promising approach to increase the predictive power of this in vitrotool and to contribute to the development of drug substances and drug products in a more biorelevant way.

Influence of temperature on microbially induced calcium carbonate precipitation for soil treatment.

Microbially induced calcium carbonate precipitation (MICP) is a potential method for improvement of soil. A laboratory study was conducted to investigate the influence of temperatures for soil improvement by MICP. The ureolytic activity experiments, MICP experiments in aqueous solution and sand column using Sporosarcina pasteurii were conducted at different temperatures(10, 15, 20, 25 and 30°C). The results showed there were microbially induced CaCO3 precipitation at all the temperatures from 10 to 30°C. The results of ureolytic activity experiments showed that the bacterial had higher ureolytic activity at high temperatures within the early 20 hours, however, the ureolytic activity at higher temperatures decreased more quickly than at lower temperatures. The results of MICP experiments in aqueous solution and sand column were consistent with tests of ureolytic activity. Within 20 to 50 hours of the start of the test, more CaCO3 precipitation was precipitated at higher temperature, subsequently, the precipitation rate of all experiments decreased, and the higher the temperature, the faster the precipitation rate dropped. The final precipitation amount of CaCO3 in aqueous solution and sand column tests at 10 °C was 92% and 37% higher than that at 30 °C. The maximum unconfined compressive strength of MICP treated sand column at 10 °C was 135% higher than that at 30 °C. The final treatment effect of MICP at lower temperature was better than that at high temperature within the temperature range studied. The reason for better treatment effect at lower temperatures was due to the longer retention time of ureolytic activity of bacteria at lower temperatures.

Approaches to increase mechanistic understanding and aid in the selection of precipitation inhibitors for supersaturating formulations - a PEARRL review.

OBJECTIVES: Supersaturating formulations hold great promise for delivery of poorly soluble active pharmaceutical ingredients (APIs). To profit from supersaturating formulations, precipitation is hindered with precipitation inhibitors (PIs), maintaining drug concentrations for as long as possible. This review provides a brief overview of supersaturation and precipitation, focusing on precipitation inhibition. Trial-and-error PI selection will be examined alongside established PI screening techniques. Primarily, however, this review will focus on recent advances that utilise advanced analytical techniques to increase mechanistic understanding of PI action and systematic PI selection. KEY FINDINGS: Advances in mechanistic understanding have been made possible by the use of analytical tools such as spectroscopy, microscopy and mathematical and molecular modelling, which have been reviewed herein. Using these techniques, PI selection can be guided by molecular rationale. However, more work is required to see widespread application of such an approach for PI selection. SUMMARY: Precipitation inhibitors are becoming increasingly important in enabling formulations. Trial-and-error approaches have seen success thus far. However, it is essential to learn more about the mode of action of PIs if the most optimal formulations are to be realised. Robust analytical tools, and the knowledge of where and how they can be applied, will be essential in this endeavour.

The application of caprylic acid in downstream processing of monoclonal antibodies.

Caprylic acid (CA), a naturally occurring eight-carbon fatty acid, has long been used as albumin stabilizer, non-IgG fraction precipitant and bactericidal agent in pharmaceutical industry. The mechanisms through which CA achieves its effects have been correlated with the molecule's protein/lipid binding capacity conferred by its octyl moiety. This article, following an initial review of CA's historical applications, introduces CA's relatively new application in downstream processing of monoclonal antibodies (mAbs). By taking advantage of CA mediated impurity precipitation and virus inactivation, it might be possible to develop a two-column purification process in replacement of the standard three-column process without compromising product quality.

Xanthine urolithiasis: Inhibitors of xanthine crystallization.

OBJECTIVE: To identify in vitro inhibitors of xanthine crystallization that have potential for inhibiting the formation of xanthine crystals in urine and preventing the development of the renal calculi in patients with xanthinuria. METHODS: The formation of xanthine crystals in synthetic urine and the effects of 10 potential crystallization inhibitors were assessed using a kinetic turbidimetric system with a photometer. The maximum concentration tested for each compound was: 20 mg/L for 3-methylxanthine (3-MX); 40 mg/L for 7-methylxanthine (7-MX), 1-methylxanthine (1-MX), theobromine (TB), theophylline, paraxanthine, and caffeine; 45 mg/L for 1-methyluric acid; 80 mg/L for 1,3-dimethyluric acid; and 200 mg/L for hypoxanthine. Scanning electron microscopy was used to examine the morphology of the crystals formed when inhibitory effects were observed. RESULTS: Only 7-MX, 3-MX, and 1-MX significantly inhibited xanthine crystallization at the tested concentrations. Mixtures of inhibitors had an additive effect rather than a synergistic effect on crystallization. CONCLUSION: Two of the inhibitors identified here-7-MX and 3-MX-are major metabolites of TB. In particular, after TB consumption, 20% is excreted in the urine as TB, 21.5% as 3-MX, and 36% as 7-MX. Thus, consumption of theobromine could protect patients with xanthinuria from the development of renal xanthine calculi. Clinical trials are necessary to demonstrate these effects in vivo.

Precipitation behavior of pioglitazone on the particle surface of hydrochloride salt in biorelevant media.

The purpose of the present study was to investigate the precipitation of a drug on the particle surface of its salt in biorelevant media. Pioglitazone (PIO, weak base, pKa = 5.8) was used as a model drug. The crystal particles of PIO hydrochloride (PIO HCl) were immobilized between two slide glasses. A biorelevant medium was penetrated between the slide glasses under a polarized light microscope. Crystalline precipitates appeared on the surface of PIO HCl within 15 s after contact with the biorelevant media. The crystalline precipitates were suggested to be a free base of PIO by microscopic Raman spectroscopy and powder X-ray diffraction. Bile micelles affected the precipitation patterns on the surface. Hydroxypropylmethylcellulose and hydroxypropylmethylcellulose acetate succinate inhibited the precipitation. The precipitation on the surface of its salt could be an important factor that could affect the dissolution profiles of a drug.

Development of a Video-Microscopic Tool To Evaluate the Precipitation Kinetics of Poorly Water Soluble Drugs: A Case Study with Tadalafil and HPMC.

Many drug candidates today have a low aqueous solubility and, hence, may show a low oral bioavailability, presenting a major formulation and drug delivery challenge. One way to increase the bioavailability of these drugs is to use a supersaturating drug delivery strategy. The aim of this study was to develop a video-microscopic method, to evaluate the effect of a precipitation inhibitor on supersaturated solutions of the poorly soluble drug tadalafil, using a novel video-microscopic small scale setup. Based on preliminary studies, a degree of supersaturation of 29 was chosen for the supersaturation studies with tadalafil in FaSSIF. Different amounts of hydroxypropyl methyl cellulose (HPMC) were predissolved in FaSSIF to give four different concentrations, and the supersaturated system was then created using a solvent shift method. Precipitation of tadalafil from the supersaturated solutions was monitored by video-microscopy as a function of time. Single-particle analysis was possible using commercially available software; however, to investigate the entire population of precipitating particles (i.e., their number and area covered in the field of view), an image analysis algorithm was developed (multiparticle analysis). The induction time for precipitation of tadalafil in FaSSIF was significantly prolonged by adding 0.01% (w/v) HPMC to FaSSIF, and the maximum inhibition was reached at 0.1% (w/v) HPMC, after which additional HPMC did not further increase the induction time. The single-particle and multiparticle analyses yielded the same ranking of the HPMC concentrations, regarding the inhibitory effect on precipitation. The developed small scale method to assess the effect of precipitation inhibitors can speed up the process of choosing the right precipitation inhibitor and the concentration to be used.

The impact of polymeric excipients on the particle size of poorly soluble drugs after pH-induced precipitation.

Active pharmaceutical ingredients (APIs) with strongly pH-dependent aqueous solubility can face the problem of precipitating from solution when the pH changes from acidic in the stomach to neutral in the intestine. The present work investigates the effect of two polymeric excipients - polyvinylpyrrolidone (PVP) and Soluplus - on the ability to either prevent precipitation, or to control the size distribution of precipitated particles when precipitation cannot be prevented. Two different APIs were compared, Dabigatran etexilate mesylate and Rilpivirine hydrochloride. The effect of excipient concentration on the precipitation behaviour during pH titration was systematically investigated and qualitatively different trends were observed: in case of Soluplus, which forms a micellar solution when critical micelle concentration is exceeded, precipitation was inhibited in the case of Dabigatran etexilate, which partitioned into the micelles. On the other hand, Rilpivirine precipitated independently of Soluplus concentration. In the case of PVP, which does not form micelles, precipitation could not be avoided. Increased polymer concentration, however prevented the aggregation of precipitated particles into larger cluster. The observed effect of PVP was especially pronounced for Rilpivirine. The main conclusion of this study is that a suitably chosen polymeric excipient can either prevent precipitation altogether or reduce the size of the resulting particles. The mechanism of action, however, seems-specific to a given molecule. It was also shown that the polymer-stabilised particles have a potential to redissolve.

Novel Displacement Agents for Aqueous 2-Phase Extraction Can Be Estimated Based on Hybrid Shortcut Calculations.

The purification of therapeutic proteins is a challenging task with immediate need for optimization. Besides other techniques, aqueous 2-phase extraction (ATPE) of proteins has been shown to be a promising alternative to cost-intensive state-of-the-art chromatographic protein purification. Most likely, to enable a selective extraction, protein partitioning has to be influenced using a displacement agent to isolate the target protein from the impurities. In this work, a new displacement agent (lithium bromide [LiBr]) allowing for the selective separation of the target protein IgG from human serum albumin (represents the impurity) within a citrate-polyethylene glycol (PEG) ATPS is presented. In order to characterize the displacement suitability of LiBr on IgG, the mutual influence of LiBr and the phase formers on the aqueous 2-phase system (ATPS) and partitioning is investigated. Using osmotic virial coefficients (B22 and B23) accessible by composition gradient multiangle light-scattering measurements, the precipitating effect of LiBr on both proteins and an estimation of both protein partition coefficients is estimated. The stabilizing effect of LiBr on both proteins was estimated based on B22 and experimentally validated within the citrate-PEG ATPS. Our approach contributes to an efficient implementation of ATPE within the downstream processing development of therapeutic proteins.

Production and characterization of poly(3-hydroxybutyrate) generated by Alcaligenes latus using lactose and whey after acid protein precipitation process.

Whey after acid protein precipitation was used as substrate for PHB production in orbital shaker using Alcaligenes latus. Statistical analysis determined the most appropriate hydroxide for pH neutralization of whey after protein precipitation among NH4OH, KOH and NaOH 10%w/v. The results were compared to those of commercial lactose. A scale-up test in a 4L bioreactor was done at 35°C, 750rpm, 7L/min air flow, and 6.5 pH. The PHB was characterized through Fourier Transform Infrared Spectroscopy, thermogravimetry and differential scanning calorimetry. NH4OH provided the best results for productivity (p), 0.11g/L.h, and for polymer yield, (YP/S), 1.08g/g. The bioreactor experiment resulted in lower p and YP/S. PHB showed maximum degradation temperature (291°C), melting temperature (169°C), and chemical properties similar to those of standard PHB. The use of whey as a substrate for PHB production did not affect significantly the final product quality.

Precipitation of sword bean proteins by heating and addition of magnesium chloride in a crude extract.

Sword bean (Canavalia gladiata) seeds are a traditional food in Asian countries. In this study, we aimed to determine the optimal methods for the precipitation of sword bean proteins useful for the food development. The soaking time for sword beans was determined by comparing it with that for soybeans. Sword bean proteins were extracted from dried seeds in distilled water using novel methods. We found that most proteins could be precipitated by heating the extract at more than 90 °C. Interestingly, adding magnesium chloride to the extract at lower temperatures induced specific precipitation of a single protein with a molecular weight of approximately 48 kDa. The molecular weight and N-terminal sequence of the precipitated protein was identical to that of canavalin. These data suggested that canavalin was precipitated by the addition of magnesium chloride to the extract. Our results provide important insights into the production of processed foods from sword bean.

The effects of apolipoprotein B depletion on HDL subspecies composition and function.

HDL cholesterol (HDL-C) efflux function may be a more robust biomarker of coronary artery disease risk than HDL-C. To study HDL function, apoB-containing lipoproteins are precipitated from serum. Whether apoB precipitation affects HDL subspecies composition and function has not been thoroughly investigated. We studied the effects of four common apoB precipitation methods [polyethylene glycol (PEG), dextran sulfate/magnesium chloride (MgCl2), heparin sodium/manganese chloride (MnCl2), and LipoSep immunoprecipitation (IP)] on HDL subspecies composition, apolipoproteins, and function (cholesterol efflux and reduction of LDL oxidation). PEG dramatically shifted the size distribution of HDL and apolipoproteins (assessed by two independent methods), while leaving substantial amounts of reagent in the sample. PEG also changed the distribution of cholesterol efflux and LDL oxidation across size fractions, but not overall efflux across the HDL range. Dextran sulfate/MgCl2, heparin sodium/MnCl2, and LipoSep IP did not change the size distribution of HDL subspecies, but altered the quantity of a subset of apolipoproteins. Thus, each of the apoB precipitation methods affected HDL composition and/or size distribution. We conclude that careful evaluation is needed when selecting apoB depletion methods for existing and future bioassays of HDL function.

Comparison of two low-density lipoprotein apheresis systems in patients with homozygous familial hypercholesterolemia.

Low-density lipoprotein (LDL) apheresis (LA) is a reliable method to decrease LDL-C concentrations and remains the gold standard therapy in homozygous familial hypercholesterolemia (HoFH). The objective of this study was to compare the efficacy of two LA systems [heparin-induced extracorporeal LDL precipitation (HELP) vs. dextran sulfate adsorption (DS) on the reduction of lipids, inflammatory markers, and adhesion molecules in a sample of genetically defined HoFH subjects (n = 9)]. Fasting blood samples were collected before and after LA. All subjects served as their own control and were first treated with the HELP system then with DS in this single sequence study. Compared with HELP, DS led to significantly greater reductions in total cholesterol (-63.3% vs. -59.9%; P = 0.05), LDL-C (-70.5% vs. -63.0%; P = 0.02), CRP (-75.3% vs. -48.8%; P < 0.0001), and TNF-α (-23.7% vs. +14.7%; P = 0.003). Reductions in the plasma levels of PCSK9 (-45.3% vs. -63.4%; P = 0.31), lipoprotein (a) (-70.6% vs. -65.0%; P = 0.30), E-selectin (-16.6% vs. -18.3%; P = 0.65), ICAM-1 (-4.0 vs. 5.6%; P = 0.56), and VCAM-1 (8.3% vs. -1.8%; P = 0.08) were not different between the two systems. For the same volume of filtered plasma (3,000 mL), however, HELP led to greater reductions in plasma apoB (-63.1% vs. -58.3%; P = 0.04), HDL-C (-20.6% vs. -6.5%; P = 0.003), and PCSK9 (-63.4% vs. -28.5%; P = 0.02) levels. These results suggest that both LA systems are effective in reducing plasma lipids and inflammatory markers in HoFH. Compared with HELP, greater reductions in lipid levels and inflammatory markers were achieved with DS, most likely because this method allows for a larger plasma volume to be filtered. J. Clin. Apheresis 31:359-367, 2016. © 2015 Wiley Periodicals, Inc.